Vanadium Stimulates the (Na+,K +) Pump Friend Erythroleukemia Cells and Blocks in Erythropoiesis

Friend murine erythroleukemia cells underwent apparently normal erythropoiesis when treated with dimethyl sulfoxide. One of the earliest events associated with this induction was a decrease in ouabain sensitive 86Rb+ uptake, an assay of the plasma membrane Na, K(ATPase). Ammonium vanadate (10/~M) blocked differentiation of these cells without affecting cell viability. Vanadium was taken up by Friend cells and prevented the dimethyl sulfoxide-induced decrease in ouabain sensitive 86Rb+ uptake. Vanadate reactivated 86Rb+ transport previously inhibited by dimethyl sulfoxide treatment but had no affect on 86Rb+ transport in untreated cells. These results suggest an essential role for the (Na,K)ATPase in cell differentiation. Friend murine erythroleukemia cells grow indefinitely in suspension but may be induced to terminally differentiate by a wide variety of drugs, including dimethyl sulfoxide (DMSO'). One of the earliest events associated with induction is a decrease in ouabain sensitive SrRb+ uptake, an assay of the plasma membrane (Na, K)ATPase (1, 2). This event appears to be the rate limiting step in commitment since pretreatment of cells with ouabain prior to adding DMSO accelerates commitment to erythropoiesis by eliminating a characteristic lag time (2). In some cells lines, ouabain alone will induce erythropoiesis (3). To further characterize the role of the (Na,K)ATPase in Friend cell differentiation we have investigated the effects of vanadate on this system. Vanadate is a potent (Na,K)ATPase inhibitor in vitro (4) but has been reported to activate this enzyme in certain hormone sensitive tissues (5). Here we show that addition of 10-20 uM ammonium vanadate to Friend cell cultures completely blocks differentiation. We also show that vanadate is taken up by the cells and reverses the DMSO induced inhibition of ouabain sensitive SrRb÷ uptake. We suggest that intracellular vanadium blocks several events essential for terminal differentiation, one of which is a decrease in the plasma membrane (Na,K) pump activity. MATERIALS AND METHODS The Friend murine erythroleukemia cells (cell line 745-PL-4 obtained from David Housman, Massachusetts Institute of Technology) were maintained at 1--40 x 104 cells/ml to insure logarithmic growth in a medium (Gibco Laboratories, Grand Island, NY) and supplemented with 13% fetal calf serum (M. A. Bioproducts, Walkersville, MD). Commitment of Friend cells to terminal ' Abbreviation used in this paper. DMSO, dimethyl sulfoxide. differentiation was assayed in plasma clot culture (6). In this technique, the cells are scored as committed to erythropoiesis if they form small colonies (<32 cells) and stain orange with benzidine. Cell viability was measured by the exclusion of trypan blue. Ouabain inhibitable ~Rb + (New England Nuclear, Boston, MA) uptake was used to assay the pumping activity of the (Na, K)ATPase as previously described (2). In this assay, suspended cells in ~Rb-labeled assay medium were centrifuged through dinonyl-phthalate/silicone oil ( 1:1) to remove extracellular radioactivity, The rate of ~Rb ÷ uptake was assumed to roughly approximate K + uptake. The uptake of ['~V]vanadate was measured using the same procedure described above. [48V]vanadyl chloride, obtained from Amersham Corp. (Arlington Heights, IL) was converted to vanadate by dilution into 100 mM NH4VO3 at pH 8.0 and incubated at 70"C for 2 h. The data were normalized to the volume of cell water estimated by the difference between the internally trapped 3H20 and the externally trapped ['4Clsucrose in the pellet in parallel experiments. Externally trapped [48V]vanadate was determined by the amount of radioactivity in the pellets of cells that were cooled to 4"C immediately before [~V]vanadate addition and centrifuged immediately afterward,